Project Summary Specific Aim 1. To identify key differentiation genes We hypothesized that epigenetic dysregulation of neural crest stem cells and/or sympathoadrenal progenitors contributes to neuroblastoma initiation, tumorigenesis and progression. Moreover, by targeting critical chromatin regulators that keep neuroblastoma in a self-renewal state we should be able to suppress growth and induce differentiation. To do this we performed an epigenetic focused siRNA screen to identify genes that control NB cell proliferation and differentiation using a high-throughput, high content imaging screen. We identified 53 candidate genes whose loss of expression results in a decrease in the number of NB cells and of these, 16 also induce morphologic and biochemical evidence of differentiation. A secondary screen using additional siRNAs excluded genes which may have resulted from off-target effects of siRNAs. Four of the candidates had already been shown to affect NB cell growth and differentiation. To prioritize those hits that would be amenable to drug development, we performed an additional screen of a tool compound library of 20 small molecule inhibitors of chromatin regulators. We evaluated the growth and differentiation in 8 NB cell lines and 2 immortal, but not transformed cell lines after exposure for 7days to 8 different drug concentrations. The secondary chemical screen identified EZH2 and SETD8 as druggable NB targets. Specific Aim 2. To characterize molecular mechanisms of action of growth and differentiation genes SETD8 is a histone methyltransferase which mono-methylates H4K20, and regulates critical cell cycle events during G1 and G2. We discovered high levels of SETD8 in primary NB tumors are associated with poor overall survival. In high-risk, Stage 4 NB patients high-levels of SETD8 are associated with poor prognosis in the MYCN-wt subgroup of high risk patients which comprises 60% of high-risk patients. We determined the mechanism of action to be due to SETD8 mono-methylation of p53 which leads to a decrease in its stability. This was the first report that genetic targeting SETD8 had activity in pre-clinical xenograft tumor models reducing tumor growth and conferring a significant survival advantage. This study was recently published in Cancer Cell 31:50-63, 2017.